Dhruve S. Jeevan

Targeting cancer stem cells in glioblastoma multiforme using mTOR inhibitors and the differentiating agent all-trans retinoic acid

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, portends a poor prognosis despite current treatment modalities. Recurrence of tumor growth is attributed to the presence of treatment-resistant cancer stem cells (CSCs). The targeting of these CSCs is therefore essential in the treatment of this disease. Mechanistic target of rapamycin (mTOR) forms two multiprotein complexes, mTORC1 and mTORC2, which regulate proliferation and migration, respectively. Aberrant function of mTOR has been shown to be present in GBM CSCs. All-trans retinoic acid (ATRA), a derivative of retinol, causes differentiation of CSCs as well as normal neural progenitor cells. The purpose of this investigation was to delineate the role of mTOR in CSC maintenance, and to establish the mechanism of targeting GBM CSCs using differentiating agents along with inhibitors of the mTOR pathways. The results demonstrated that ATRA caused differentiation of CSCs, as demonstrated by the loss of the stem cell marker Nestin. These observations were confirmed by western blotting, which demonstrated a time-dependent decrease in Nestin expression following ATRA treatment. This effect occurred despite combination with mTOR (rapamycin), PI3K (LY294002) and MEK1/2 (U0126) inhibitors. Expression of activated extracellular signal-regulated kinase 1/2 (pERK1/2) was enhanced following treatment with ATRA, independent of mTOR pathway inhibitors. Proliferation of CSCs, determined by neurosphere diameter, was decreased following treatment with ATRA alone and in combination with rapamycin. The motility of GBM cells was mitigated by treatment with ATRA, rapamycin and LY29002 alone. However, combination treatment augmented the inhibitory effect on migration suggesting synergism. These findings indicate that ATRA-induced differentiation is mediated via the ERK1/2 pathway, and underscores the significance of including differentiating agents along with inhibitors of mTOR pathways in the treatment of GBM.

Cerebrospinal Fluid Leaks and Encephaloceles of Temporal Bone Origin: Nuances to Diagnosis and Management

OBJECTIVE Temporal bone encephalocele has become less common as the incidence of chronic mastoid infection and surgery for this condition has decreased. As a result, the diagnosis is often delayed, and the encephalocele is often an incidental finding. This situation can result in serious neurologic complications with patients presenting with cerebrospinal fluid leak and meningitis. We review the occurrence of, characteristics of, and repair experience with temporal encephaloceles from 2000-2012. METHODS We conducted a retrospective review of 32 patients undergoing combined mastoidectomy and middle cranial fossa craniotomy for the treatment of temporal encephalocele. RESULTS The diagnosis of temporal encephalocele was made in all patients using high-resolution temporal bone computed tomography and magnetic resonance imaging. At the time of diagnosis, 12 patients had confirmed cerebrospinal fluid leak; other common presenting symptoms included hearing loss and ear fullness. Tegmen defect was most commonly due to chronic otitis media (n = 14). Of these patients, 8 had undergone prior mastoidectomy, suggesting an iatrogenic cause. Other etiologies included radiation exposure, congenital defects, and spontaneous defects. Additionally, 2 patients presented with meningitis; 1 patient had serious neurologic deficits resulting from venous infarction. CONCLUSIONS The risk of severe neurologic complications after the herniation of intracranial contents through a tegmen defect necessitates prompt recognition and appropriate management. Computed tomography and magnetic resonance imaging aid in definitive diagnosis. A combined mastoid/middle fossa approach allows for sustainable repair with adequate exposure of defects and support of intracranial contents.

Hemorrhagic Ganglioglioma of the Posterior Fossa: Case Report

Gangliogliomas are rare tumors of the central nervous system that are usually found in the supratentorial compartment, although cases throughout the nervous system have been described. They are generally low-grade malignancies that are amenable to cure by surgical resection. Most manifest as seizures, though, based on location, they can present with focal neurological deficits. We present here a rare case of an infratentorial ganglioglioma presenting with hemorrhage. To our knowledge this is the only reported case of a hemorrhagic ganglioglioma and, as such, we examine its possible prognosis.

Malignant carotid body tumor presenting with myelopathy: case report.

Malignant carotid body tumors are rare, with spread of the tumor mostly noted in regional lymph nodes. Vertebral metastases are an exceedingly rare presentation, only reported in isolated case reports, and present a diagnostic and management challenge. A case of widespread vertebral metastasis, presenting with myelopathy, from a carotid body tumor is discussed in this paper, along with management strategies.

Abstract 4358: Involvement of mTOR pathway in metastatic brain tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Metastatic dissemination to the brain occurs in almost forty percent of all cancers, and is ten times more common than primary brain tumors. Mammalian target of Rapamycin (mTOR), an atypical PI3K related kinase, exists in two distinct multiprotein complexes (mTORC1 and mTORC2). mTORC1/2 regulate various cellular functions and are shown to be activated in primary brain tumors such as glioblastoma and medulloblastoma. In addition, metastatic tumor cells possess a unique reprogramming resulting in a stem cell like phenotype with invasive, migratory and proliferative potential similar to that seen in glioblastoma, where mTOR appears to play an essential role. However, the involvement of the mTOR pathway in metastatic brain tumors remains to be elucidated. This study aimed to test the hypothesis that the mTOR pathway plays a critical role in metastatic brain tumors. In order to achieve our goals, expression of mTOR and its components (Raptor and Rictor) were studied in metastatic brain tumors using immunohistochemical analysis. Further, the function of mTOR in relation to proliferation, migration, and cell spreading was studied in a metastatic breast cancer cell line (MBA-MD 231). The expression of epithelial marker E-Cadherin and meschenchymal marker Vimentin following the inhibition of mTOR using pharmacological inhibitors (Rapamycin and PP242) as well as short interfering RNA (mTOR, Raptor and Rictor) was examined by immunofluorescence analysis. Results demonstrated that a significant number of metastatic brain tumors expressed mTOR, Raptor, and Rictor; 50% expressing all three components. The metastatic potential showed that migration of cancer cells was inhibited significantly by mTORC1 inhibition (Rapamycin) and mTORC1/2 inhibition (PP242) by 29% and 64%, respectively. Utilizing the EdU incorporation technique, S-phase cell cycle entry analysis rendered a 52% decline in proliferation after Rapamycin treatment. Cell spreading/attachment analysis exhibited total attachment of control cells within 20 minutes of plating. This effect was prevented by pretreatment with Rapamycin or PP242. Immunoflourescence analysis showed that E-cadherin underwent forced nuclear localization following acute inhibition of mTOR, using Rapamycin or siRNA treatments. These results were substantiated by Western blotting, suggesting its role in cellular reprogramming, namely Epithelial-Mesenchymal Transition or its counterparts. However, Vimentin expression remained unaltered. In conclusion, these results provide evidence that mTOR is enhanced in metastatic brain tumors, suggesting its critical role in achieving a metastatic potential to the brain. Importantly, these findings underscore the therapeutic value of mTOR inhibition in the management of patients with metastatic brain tumors. Citation Format: Amanda Kwasnicki, Dhruve Jeevan, Anita Goyal, Alex Braun, Raj Murali, Meena Jhanwar-Uniyal. Involvement of mTOR pathway in metastatic brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2013-4358

Abstract 273: Molecular markers promoting metastases to the brain via EMT: Genes, proteins, and functional analysis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Brain metastasis is the most common cause of mortality and morbidity of many cancers and occurs in more than 40% of patients. The metastatic cascade entails an orderly sequence of steps enabling tumor cells to migrate from the primary tumor and colonize at secondary locations. The underlying mechanisms of metastatic dissemination to the brain remain to be defined. However, In order to achieve this metastatic potential a cancer cell may go through a cellular reprogramming process, namely epithelial-to-mesenchymal transition (EMT), and its counterpart mesenchymal-to-epithelial transition (MET), by acquiring multiple genetic alterations and a stem cell like phenotype. We hypothesized that EMT would play a crucial role in brain metastasis. In order to achieve our goal we studied the expression of markers of EMT, MET, and stem cells in metastatic brain tumor samples. Microarray analysis of metastatic tumors was also done using a plate form of 17,000 genes. Furthermore, functional analysis establishing the metastatic phenotype of cancer cells towards a cerebral environment was also demonstrated using metastatic cell lines. Our results demonstrated that the expressions of EMT markers (Snail, TWIST, pSTAT3, NFkB), as well as the mesenchymal marker Vimentin, were present in metastatic tumor samples. The stem cell marker CD44 was also highly expressed. Gene expression analysis established the high presence of transcriptional factors associated with differentiation and reprogramming. Lastly, Immunofluorescence analysis demonstrated the presence of the mesenchymal marker Vimentin in brain tumor specimens. Tumor cells grown in astrocytic media displayed an increased cell proliferation, as well as enhanced S-phase cell cycle entry using an EDU incorporation technique. Migration of primary tumor cells was significantly enhanced in astrocytic media as evidenced by scratch wound migration. Furthermore, the tumor cells have a greater affinity for astrocytic media as demonstrated by chemotatic migratory tests. Moreover, co-culture analysis of primary tumor cells with astrocytic cells shows amicable mutual growth with Immunofluorescence demonstrating cell-to-cell interaction via E-Cadherin. In conclusion results of this study suggests that reprogramming via EMT/MET plays a crucial step in brain metastasis, and that the cerebral milieu provides a suitable microenvironment for metastatic cells to grow and disseminate. Citation Format: Dhruve S. Jeevan, Sudeepta Sridhara, Alex Braun, Raj Murali, Meena Jhanwar-Uniyal. Molecular markers promoting metastases to the brain via EMT: Genes, proteins, and functional analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 273. doi:10.1158/1538-7445.AM2013-273